The enzyme deoxythymidine kinase (dTk) provides the eucaryotic cell with a means for utilizing deoxythymidine (dT), which is not an intermediate in the thymidylate synthesis de novo. For this reason dTk is considered a salvage enzyme, introducing dT into the DNA metabolism. As the major dTk form of mammalian cell is only present during cell division, the dTk has been denominated scavenger enzyme.
Three different cellular isoenzymes in human cells have been described. The cytosolar dTk, called dTk-F, which occurs in optimal amounts in dividing cells (stages Gl to S) (Bello, Exptl. Cell Res. 89: 263, 1974; Littlefield, Biochim. Biophys. Acta. 115: 398, 1966) and is more or less absent in resting cells. In humans this enzyme is coded for in chromosome 17 near the galactokinase locus. The second cellular isoenzyme is the mitochondrial, denominated dTk-A, which is present in the mitochondrial matrix. The activity of this dTk remains relatively constant during the different cell stages (Adelstein et al; Develop. Biol. 216: 537, 1971), and dTk-A is coded for by chromosome 16. The third dTk, a minor activity called dTk-B, has only been reported in continuous cell lines HeLa and KB and is said to be confined to the inside of the mitochondrial membrane (reviewed by Kit, Pharmacol. Ther. 4: 501, 1979).
The three cellular dTks differ in biochemical properties. The dTk-F and dTk-B, which are quite similar, are distinguished, besides as to localization, by isoelectric focusing, having different pI, and by electrophoretic mobility. In contrast to dTk-F and dTk-B, the dTk-A accepts cytidinetriphosphate (CTP) as a phosphate donor and is not as sensitive as the others to dTTP (deoxythymidinetriphosphate) feedback inhibition. The dTk-A also phosphorylates deoxycytidine (dC) and is inhibited by dCTP (reviewed by Kit, Pharmacol. Ther. (4: 501, 1979).
With regard to viruses, specific isoenzymes, coded by the viral genome, have been shown in the cell after infection with viruses from the Herpes group and the Pox group. Enzymatically the human virus specific dTks resemble dTk-A, except for the vaccinia dTk which cannot utilize CTP as phosphate donor and not either can phosphorylate deoxycytidine. This dTk is easily distinguished from the human cellular dTks by electrophoresis (Kit et al., Progr. Med. Virol. 21: 13, Karger Basel 1975). Both the HSV dTks and the VZV dTk have a broader spectrum of possible phosphate donors and accept different pyrimidines and pyrimidine analogues as substrates (Cheng et al, Biochim. Biophys. Acta. 452: 370, 1976, and J. Virol. 31: 172, 1979). The competitive blocking of dTk isoenzyme mediated dT conversion to dTmp (deoxythymidinemonophosphate) exerted by the dT-analogue 2'-deoxy-5-iodourine (IUdR), has been known for a long time. The use of radio-labeled IUdR directly as a substrate to gain high sensitivity in assays of viral dTks was shown by us (Gronowitz & Kallander, Infec. Immun. 29: 425, 1980).
As mentioned dTk-F occurrence in cells is coupled to cell proliferation, and it is more or less absent in the differentiated cell (Munch-Petersen & Tyrsted, Biochim. Biophys. 478: 364, 1977). Studies of dTk activity in transplantable mouse tumours have revealed high dTk activities with correlation to growth rate (Bresnick et al, Cancer Res. 29: 1969, and Cancer Res. 31: 743, 1971). Recent reports have demonstrated enhanced dTk-F in peripheral blood lymphocytes of some patients suffering from malignant non-Hodkin's lymphoma and lymphatic leukemia (Ellims et al, Cancer Res. 41: 691 and Brit. J. Haematol. 49: 479, 1981). These researchers have also shown enhanced serum dTk levels in some patients from the non-Hodkin's group (Ellims et al, Blood 58: 926, 1981). Due to the conventional dTk assay used, employing .sup.3 H-dT in high concentration (5.times.10.sup.-6 M), dTk activity could only be found in a few patients with advanced disease. Further the dTk activity could be evaluated as a prognostic marker only if differential analysis regarding dTk-F and dTk-A was performed.
Conventionally dTk activity has thus been measured by following the conversion of either .sup.3 H- or .sup.14 C-labeled dT to dTmp with ATP as the phosphate donor. We have recently designed an improved dTk assay system for viral dTk isoenzymes, which uses .sup.125 I-Iododeoxyuridine (IUdR) as the substrate. The improved sensitivity obtained allowed the detection of dTk from as little as 25 HSV-infected cells. In contrast the conventional assay based on the use of .sup.3 H-dT, at a normally used concentration of 10.sup.-5 M, would require at least 450 times more enzyme. However, even though being useful in studies of viral dTks this improved assay system was not appropriate for long time assays of cellular dTk-F, partly due to the instability of this enzyme. The inability of this method of detecting the presence of minute dTk amounts in serum is easily extracted from the studies over occurrence of virus dTk blocking antibodies (dTk-ab). In these studies more than 275 human sera were assayed for dTk-ab but disturbing serum dTk activities were found in only two cases (Gronowitz & Kallander, Infec. Immun. 29: 425, 1980; Gronowitz & Kallander, J. Med. Virol. 8: 177, 1981; Kallander et al, Infec. Immun. 36: 30, 1982).